Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters in trans

One type of homology-dependent gene silencing in transgenic plants involves a silencing locus that is able to transcriptionally inactivate and methyla te an unlinked target locus with which it shares sequence identity in promo ter regions. In a manner resembling paramutation of endogenous genes, the t arget locus reactivates and loses methylation progressively over several ge nerations after segregating away from the silencing locus, which autonomous ly acquires stable methylation. To investigate the origins of trans-silenci ng ability and susceptibility, we have analyzed the structures, flanking DN A sequences and chromosomal locations of a nopaline synthase promoter silen cing locus, H-2,and a sensitive target locus, K-81 A partially resistant ta rget locus, K-alpha has been characterized molecularly. The complex and scr ambled H-2 locus comprises six copies of the nopaline synthase promoter, tw o of which are collinear with prokaryotic non-T-DNA sequences, and is integ rated close to a region of intercalary heterochromatin. These features prob ably contribute collectively to the silencing ability because H-2 subclones reintroduced into random locations in the K-81 genome did not frequently i nduce silencing. Both the K-81 and K-alpha loci have simple structures, alt hough the former contains non-T-DNA prokaryotic sequences that are also pre sent at H-2, and they are flanked by low copy plant DNA. H-2 and K-81 might interact effectively because they are present on morphologically similar c hromosomes from the T sub-genome of allotetraploid tobacco.